Identification of an immunogenic subset of metastatic uveal melanoma · Identification of an immunogenic subset of metastatic uveal melanoma Luke D. Rothermel1, Arvind C. Sabesan1,

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Identification of an immunogenic subset of metastatic uveal melanoma Luke D. Rothermel*1, Arvind C. Sabesan*1, Daniel J. Stephens*1, Smita S. Chandran1, Biman C. Paria1, Abhishek K. Srivastava1, Robert Somerville1, John R. Wunderlich1, Chyi-Chia R. Lee2, Liqiang Xi2, Trinh H. Pham2, Mark Raffeld2, Parthav Jailwala3, Manjula Kasoji3, and Udai S. Kammula*1

* Contributed equally as first author 1 Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 2 Laboratory of Pathology, National Cancer Institute, Bethesda, MD National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 3 Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research (FNLCR), Leidos Biomedical Research Inc., Frederick, MD 21702 Running title: Identification of an immunogenic subset of metastatic uveal melanoma Key Words: Uveal melanoma, cutaneous melanoma, T cells, liver metastases Disclosures: There are no commercial or financial disclosures. Number of figures/ tables: 5/2 Number of references: 50 Correspondence: Udai S. Kammula, MD Surgery Branch, Center for Cancer Research National Cancer Institute 10 Center Drive Building 10-Hatfield CRC, Rm 3-5930 Bethesda, MD, 20892-1201 Tel: 301-435-8606 Fax: 301-435-5167 Email: [email protected]

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Statement of Translational Relevance

Although remarkable strides have been achieved in the management of metastatic cutaneous

melanoma (CM) with T cell based immunotherapies, limited progress has been made with

metastatic uveal melanoma (UM), a rare and aggressive variant that is hypothesized to be

immunotherapy-resistant. In this study, we sought to formally define the relative

immunogenicity of these two melanoma variants and determine whether endogenous anti-tumor

immune responses exist against UM. Here, we report the novel identification of TIL from a

subset of UM metastases with robust anti-tumor reactivity, comparable in magnitude to that of

CM TIL. The discovery of this immunogenic group of UM metastases has important clinical

implications for the role of immunotherapies in the treatment of patients who harbor these unique

tumors.




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ABSTRACT

Purpose: Uveal melanoma (UM) is a rare melanoma variant with no effective therapies once

metastases develop. Although durable cancer regression can be achieved in metastatic cutaneous

melanoma (CM) with immunotherapies that augment naturally existing anti-tumor T cell

responses, the role of these treatments for metastatic UM remains unclear. We sought to define

the relative immunogenicity of these two melanoma variants and determine whether endogenous

anti-tumor immune responses exist against UM.

Experimental Design: We surgically procured liver metastases from UM (n=16) and CM

(n=35) patients and compared the attributes of their respective tumor cell populations and their

infiltrating T cells (TIL) using clinical radiology, histopathology, immune assays and whole

exomic sequencing.

Results: Despite having common melanocytic lineage, UM and CM metastases differed in their

melanin content, tumor differentiation antigen expression, and somatic mutational profile.

Immunologic analysis of TIL cultures expanded from these divergent forms of melanoma

revealed CM TIL were predominantly composed of CD8+ T cells, while UM TIL were CD4+

dominant. Reactivity against autologous tumor was significantly greater in CM TIL compared to

UM TIL. However, we identified TIL from a subset of UM patients which had robust anti-tumor

reactivity comparable in magnitude to CM TIL. Interestingly, the absence of melanin

pigmentation in the parental tumor strongly correlated with the generation of highly reactive UM

TIL.

Conclusions: The discovery of this immunogenic group of UM metastases should prompt

clinical efforts to determine whether patients who harbor these unique tumors can benefit from

immunotherapies that exploit endogenous anti-tumor T cell populations.




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INTRODUCTION

Uveal melanoma (UM) is a rare and aggressive variant of melanoma that has specific

origin within the vascular layers of the eye including the choroid, ciliary body, and iris

(collectively known as the uvea) (1). Although UM is the most common intraocular tumor in

adults, it accounts for only 3% of all melanomas (2). With an annual incidence of 5.1 per million

in the U.S, UM is significantly less common than cutaneous melanomas (CM). Interestingly, UM

and CM have a shared lineage, with each arising from neural crest derived melanocytes that are

resident to their respective tissues of origin (3). Both forms of melanoma, consequently, share

prominent expression of prototypic melanocytic differentiation antigens (MDAs) such as

MART-1, gp100, and tyrosinase (4-6). Despite these similarities, UM can be distinguished from

CM by characteristic cytogenetic changes (7) and an unusual predilection to primarily

metastasize to the liver (1). Further, there exists a striking dichotomy between the clinical

management of patients with advanced UM and CM. Immunotherapies have become the main

treatment modality for metastatic CM based upon substantial evidence that tumor antigens

expressed by CM can be vigorously recognized by T cell populations endogenous to the host

immune system (8). By clinically augmenting these immune responses with either systemic

cytokines (9), antibodies targeting T cell checkpoint molecules (10, 11), or adoptive transfer of

autologous tumor infiltrating lymphocytes (TIL) (12), significant and potentially curative cancer

regression can now be achieved in advanced CM patients. However, the role of these immune

based therapies for the treatment of metastatic UM remains unclear. Patients with UM are

frequently excluded from metastatic melanoma immunotherapy clinical trials because UM is

generally thought to be an immunotherapy resistant subtype of melanoma. It has been speculated

that since the primary tumor arises in the eye, an immune privileged site, the tumor and its




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metastases harbor local immunosuppressive or cellular immuno-evasive factors that render

immunotherapies unsuccessful (13-16). Another theory proposes that since UM tumors have far

fewer somatic mutations compared to sun-exposed CM tumors (17), there are consequentially

fewer potential mutated neo-epitope targets for effective anti-tumor immunity. The poor

immunogenicity of UM has been further suggested based upon the comparatively low response

rates seen in UM patients enrolled into small pilot trials of immune modulating agents such as

interleukin-2 (18) and anti-CTLA-4 antibody (19-21). Collectively, these observations have

fostered the prevalent belief that UM, in distinction to CM, is a non-immunogenic form of

melanoma. However, this hypothesis has largely been based upon inference without formal

comparative studies performed directly upon UM and CM metastases to accurately assess their

relative immunogenicity. In this study, we aimed to address this deficiency by comparing tumor

antigen expression, tumor mutational load, and endogenous anti-tumor immunologic reactivity

found in fresh surgically resected UM versus CM metastases. By determining whether tumor

specific immune responses naturally exist against UM metastases, we sought to provide insight

into the management of this rare melanoma variant with immunotherapies that can exploit these

endogenous T cell populations.




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METHODS

Study population

A retrospective review of a prospectively maintained database identified 49 patients who

underwent liver metastatectomy with a diagnosis of metastatic melanoma at the Surgery Branch

of the National Cancer Institute between 2004 and 2014. All patients signed an institutional

review board approved consent for tumor tissue procurement and participation in subsequent

immunotherapy protocols if the patient required further systemic therapy. Inclusion criteria

included pathologically confirmed melanoma, 16 years of age or older, negative serology for

HIV, Hepatitis B and C, good performance status (Eastern Cooperative Oncology Group ≤2) and

life expectancy greater than 3 months. Patients were stratified into two cohorts based upon the

anatomic origin of their primary melanoma. The cutaneous melanoma (CM) cohort included 35

patients; 33 of these patients had documented primary tumors arising from the cutaneous

epithelium and 2 additional patients had primary tumors of unknown origin. The uveal

melanoma (UM) cohort included 14 patients who had ophthalmologic documentation that their

primary melanoma tumors arose specifically from the uveal tract. Patients with documented

primary tumors arising from mucosal and conjunctival sites were excluded from analysis.

Tumor procurement

Patients typically underwent resection of a single metastatic liver deposit or a closely

approximated cluster of tumors using standardized hepatobiliary surgical techniques.

Immediately upon resection, the fresh tumor underwent pathologic assessment, dissection, and

processing in the Surgery Branch Cell Production facility in conjunction with a clinical surgical

pathologist and research staff. Tumor tissue was assigned a unique liver metastasis identification




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number (ID #) and allocated for gross and histopathologic analysis, mutational analysis, and TIL

culture establishment using methods as described below. Although the main study exclusively

focused upon liver metastases, a set of extrahepatic metastases from 8 additional UM patients

were incorporated into the tumor driver mutational analysis, as described below.

In situ MRI assessment of tumor melanin content

All patients underwent pre-operative MRI liver imaging as part of their radiographic tumor

staging. Quantitative T1-weighted signal intensity measurements (without gadolinium

enhancement) of the in situ liver metastases and adjacent normal tissue were obtained using

clinical radiology imaging software (Carestream Vue Solutions, version 11.3). Mean tumor and

normal intensity were calculated by averaging three separate signal intensity measurements.

Hyperintense tumors were defined as having a mean tumor/normal (T/N) intensity ratio > 1.5.

Hypointense tumors had a mean T/N ratio < 0.7. Mixed intensity tumors had both hyperintense

and hypointense components. The T/N signal intensity ratio for each liver metastasis was

objectively calculated for each metastasis and scored as either hyperintense (2+), mixed intensity

(1+), or hypointense (0), as illustrated in Supplementary Figure 1.

Gross pathologic assessment of tumor melanin pigmentation

After surgical resection, all liver metastases underwent independent gross pathological

assessment and photo documentation by a board certified pathologist who was blinded to the

comparative analysis. Each metastasis underwent serial sectioning to assess their melanin

pigmentation. Tumors were scored based on their level of pigmentation as either hyperpigmented

(2+), mixed pigmented (1+), or hypopigmented (0).




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Immunohistochemical staining analysis of tumor metastases

Surgically resected tumor specimens were fixed in 10% neutral buffered formalin for up to 24

hours and routinely processed. Paraffin-embedded tissue sections of 5 mm were deparaffinized

through xylene and graded series of alcohols. Immunohistochemical staining was performed

following heat-induced epitope retrieval with target retrieval solution (low pH; DAKO,

Carpinteria, CA). Slides were incubated in Tris with 3% goat serum for 15 minutes and then

incubated at room temperature with primary antibody for 1 to 2 hours. Immunohistochemical

stainining was carried out using the Dako Autostainer or Ventana BenchMark XT Slide Stainer

(for CD3 antibody) using manufacture supplied reagents and standard protocols with the

following primary antibodies: MART-1 (no. CMC756, 1:200; Cell Marque, Rocklin, CA);

HMB45 (no. 30930, 1:4; Enzo Life Sciences, Farmingdale, NY); Tyrosinase (no. NCL-TYROS,

1:20; Novocastra Division, Leica Microsystems, Buffalo Grove, IL); MHC Class I (HC-10,

1:1000; provided by Dr. Soldano Ferrone); HLA-DR (TAL.1B5, 1:200; DAKO); CD20 (L26,

1:500; DAKO); CD8 (CD8/144B, 1:50; DAKO); CD4 (1F6, 1:80; Novacastra); CD3 (2GV6,

prediluted; Ventana). Detection was carried out using an automated slide stainer (Autostainer;

DAKO) with either horseradish peroxidase/3,3′-diaminobenzidine polymer-based detection

system (Envision+; DAKO) or a red chromogen (Liquid Permanent Red Substrate-Chromogen;

DAKO) for darkly pigmented tumors. The immunohistochemical staining was prospectively

assessed and quantitated by 2 board certified pathologists who were blinded to the comparative

analysis of the study. The percentage of viable tumor cells expressing a given marker was

quantified as 0-5%, 6-50%, or >50%. Staining intensity for each marker was graded on a scale of

0 (no staining), 1+, 2+, or 3+ (high intensity staining). Lymphocytic infiltrate was assessed with

CD4, CD8, CD3, and CD20 staining and quantified based upon the percentage of tumor




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occupied by infiltrating lymphocytes as 0 (no lymphocytes detected), 1+ (<5% of tumor field),

2+ (5-50% of tumor field), or 3+ (extensive lymphoid aggregation occupying over 50% of the

tumor field).

Generation and assessment of TIL cultures

Geographically discrete 1 to 2 mm3 tumor fragments (n=24) were freshly dissected from each

tumor metastasis and placed individually in wells of a 24-well culture plate containing complete

media with human AB serum and recombinant IL-2 (3000IU/ml) as previously described(22).

After approximately 2 weeks of culturing, each of the wells was assessed for successful TIL

expansion based upon cell count and visual inspection. Expanded TIL cultures underwent flow

cytometric phenotypic analysis after staining with anti-human CD3, CD8, and CD4 monoclonal

antibodies and their respective isotype controls (BD Biosciences). Immunofluorescence,

analyzed as the relative log fluorescence of live cells, was measured using a FACSCanto II flow

cytometer with FACSDiva software (BD Biosciences) and FlowJo software (Tree Star, Inc.).

The specific anti-tumor reactivity of individual TIL cultures was assessed by co-culture with

autologous tumor digest which had been freshly cryopreserved at the time of surgical

procurement. Briefly, TIL cells (1x105 cells) and autologous tumor digest (1x105 cells) were co-

incubated in a 0.2-ml volume in individual wells of a 96-well plate. Supernatants were harvested

from duplicate wells after 20–24 hours and IFN-γ secretion was measured in culture supernatants

using commercially available IFN-γ ELISA kits (Endogen). All data is presented as a mean of

duplicate samples. Cultures with IFN-γ production greater than 100 pg/ml and twice background

of unstimulated TIL and autologous tumor digest alone were considered as having specific anti-

tumor reactivity.




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Whole exomic sequencing and driver mutational analysis

Exome libraries were prepared from paired UM metastasis and normal samples using Agilent

SureSelectXT Human All Exon V5+UTR target enrichment kit as per the manufacturer’s

protocols (Agilent, Santa Clara, USA). The samples were pooled 3 samples per lane and

sequenced on an Illumina HiSeq2000 sequencer with TruSeq V3 chemistry (paired-end, 101bp

read length). Basecalling was carried out using Illumina RTA 1.12.4.2 run-time analysis software

and demultiplexing was carried out using Casava 1.8.2. Each sample had >99 million pass

filtered reads, with >92% bases having a base quality value >Q30 (Q30: The percentage of bases

called with an inferred accuracy of 99.9% or above, a measure of basecalling quality). The

percentage of unique library fragments was >90% across all samples. The capture efficiency as

measured by the percentage of the reads mapping on the target regions, was >60%. The mean

coverage on target regions for all samples was between 60X to 90X with >82.9% of the target

regions having at least 30X coverage. The quality of the raw reads was assessed using

FastQC(23) and NGSQCtoolkit(24). Reads were trimmed and filtered for adapters using

Trimmomatic(25). Alignment was carried out to the human Hg19 reference sequence using

BWA-0.7.4(26). Alignment files were indexed, sorted and duplicates were removed. Re-

alignment around InDels and base-quality score recalibration was carried out as per the GATK

best practices for exome-seq analysis(27). MuTect was used in the high-confidence (HC) mode

for calling somatic point mutations(28). The subset of calls that passed the high-confidence

filters after the statistical analysis within Mutect were annotated using Annovar(29) to find both

the location and the functional significance of the mutations. Mutations were filtered to keep

only those that had Mutation_info: exonic or splicing only; Consequence: non-synonymous or

stopgain_SNV. To serve as a reference group, WES data was obtained for 278 CM metastases




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via the “Skin Cutaneous Melanoma” (SKCM) data portal of The Cancer Genome Atlas (TCGA).

Somatic mutation counts for the metastatic patient samples for the SKCM cohort were extracted

from the Level 2 MAF file downloaded off the GDAC Firehose resource(30). As only somatic

point mutations were of interest while comparing the distribution of somatic mutations between

CM and UM cohorts, InDel calls (1.2%) were removed from the TCGA-SKCM cohort. As the

annotations for the point mutations in the TCGA-SKCM cohort did not have the same

terminology as for the UM cohort, the SKCM mutations were re-annotated using Annovar, with

the same filters applied based on the combination of ‘Consequence’ and ‘Mutation_info’

columns. The frequency among the CM and UM tumors for specific mutations in BRAF, GNAQ,

and GNA11 was determined primarily from WES analysis. As validation in selected samples,

library preparation with 10-20ng genomic DNA was performed using Ion AmpliSeq Cancer

Hotspot, Panel V2 and Ion AmpliSeq Library Kit 2.0, using the corresponding User Guide (Life

Technologies). The amplicon panel includes 207 primer sets covering ~2,800 COSMIC hotspot

mutations in 50 genes. Next generation sequencing was performed in an Ion Torrent Personal

genome machine (PGM), and analyzed with Torrent Suite Software (Life Technologies).

Annotation and interpretation of all variants were performed in Ion Reporter that links to

multiple databases such as RefSeq, OMIM, Oncomine, COSMIC, and dbSNP. Reported

mutations were confirmed by inspection of alignments using the Integrative Genomics

Viewer(31).

Statistical Analysis

Fisher’s exact test was used to determine associations between dichotomous demographic

parameters and the Wilcoxon rank sum test was utilized for the comparison of continuous

parameters such as patient age. Non-parametric comparisons between the UM and CM cohorts




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were performed with the Mann-Whitney test. Student’s t test was used to compare the means of

parametric variables. Linear regression analysis was used for correlation studies and presented as

R2 values. All P values are 2-tailed and have not been adjusted for multiple comparisons. In view

of the exploratory analyses performed, p<0.05 would be considered statistically significant while

0.05 < p <0.1 would be considered a trend. Excel and GraphPad Prism (v6.01) were used for

analyses.




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RESULTS

Patient demographics and procurement of liver metastases

Between 2004 and 2014, a total of 49 patients with metastatic melanoma underwent liver

metastasectomy in the context of approved clinical trials in the Surgery Branch, NCI. The current

study selectively analyzed liver metastases because of our previous finding that human

melanoma metastases demonstrate significant heterogeneity in tumor antigen expression and

lymphocytic infiltrate when stratified based upon their anatomic location in the body (6).

Further, since UM predominantly metastasizes to the liver, this homogeneous source of

metastases would prevent potential site-specific bias in our comparative assessment of tumors.

Patients undergoing liver metastasectomy were stratified into two cohorts, CM and UM, based

upon the anatomic origin of their primary melanomas. The CM cohort included 35 patients; 33 of

whom had documented primary tumors arising from the cutaneous epithelium and 2 additional

patients had primary tumors of unknown origin. Patients with melanoma of unknown origin were

included in the CM cohort based upon recent molecular genetic studies which revealed these

tumors strongly resembled cutaneous melanomas (32). The UM cohort included 14 patients who

had ophthalmologic documentation that their primary melanoma arose specifically from the

uveal tract. The characteristics for each of the patients who underwent liver metastasectomy are

shown in Table 1 and the comparison of the CM and UM cohorts are shown in Table 2. The age

(mean and range) and gender distribution of the patients in the two cohorts were similar. At the

time of referral to our center, there was a greater trend for the UM patients to have not received

prior systemic therapy for their metastatic disease when compared to the CM patients (UM: 71%

vs. CM: 37%, P= 0.06). This finding likely reflected the growing availability of approved

systemic agents and clinical trial opportunities for patients with metastatic CM during the study




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period. Of note, 63% of the CM patients had undergone prior systemic immunotherapy, whereas

only 21% of UM patients had received such treatments (CM vs. UM, P=0.01). Although both

melanoma cohorts demonstrated metastatic spread to extrahepatic sites, the UM patients had

metastases more often confined to the liver (UM: 43% vs. CM: 14%, P= 0.05), while the CM

patients demonstrated a trend toward more frequent metastases to lymph nodes and soft tissues

(CM: 60% vs. UM: 29%, P= 0.06). For surgical tumor procurement, patients typically underwent

resection of a single metastatic liver deposit or a closely approximated cluster of tumors. The

size (mean/median) of the tumor deposits resected from the patients were not significantly

different between the groups (CM: 6.2/6.0cm vs. UM: 7.2/5.5cm, P= 0.50). The cumulative

metastatic tissue that was procured at operation was assigned a unique liver metastasis

identification number (ID #) for subsequent analysis. Two UM patients (1 and 3) developed

metachronous liver metastases during the study period. These patients underwent two

independent liver metastasectomy operations and their individual tumor procurements were

assigned unique liver metastasis ID #. Thus, in sum, there were 35 CM liver metastases and 16

UM liver metastases that were available for direct comparative analysis.

Radiographic and pathologic comparison of the melanin content between CM and UM

liver metastases

Both CM and UM primary tumors arise from transformed melanocytes. Yet, despite this

common origin from melanin-producing cells, the metastases of these tumors can display

significant heterogeneity in the quantitative expression of prototypic melanin associated proteins

(6). These prior findings prompted us to ask whether CM and UM metastases demonstrated

fundamental differences in their melanin pigmentation. To address this question, we utilized pre-

operative clinical radiographic imaging and post-operative gross pathologic examination to




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evaluate the melanin content found in the liver metastases procured from CM and UM patients.

A prior study characterizing melanoma metastases with clinical MRI imaging found the in situ

tumor signal intensity from T1-weighted sequences strongly correlated with the degree of

melanin pigmentation found in those tumors after resection (33). For the current study, we

utilized these defined MRI parameters to perform in situ characterization of 30 liver metastases

identified in CM patients and 16 liver metastases in UM patients. Quantitative T1-weighted

signal intensity measurements (without gadolinium enhancement) of the in situ tumor and

adjacent normal tissue were obtained using clinical radiology imaging software (Carestream Vue

Solutions, version 11.3). The normalized tumor signal intensity (relative to normal liver) was

objectively calculated for each metastasis and scored as either hyperintense (2+), mixed intensity

(1+), or hypointense (0), as illustrated in Supplementary Figure 1. Additionally, after surgical

resection, metastases underwent independent gross pathologic examination and tumor

pigmentation was visually scored as either hyperpigmented (2+), mixed pigmented (1+), or

hypopigmented (0). The comparison of pre-operative in situ MRI intensity scoring and post-

operative pathologic pigmentation scoring for representative liver metastases is shown in Figure

1A. Analysis of the entire population of liver metastases (n=46) revealed a strong and direct

correlation between the MRI and the pathologic scoring of pigmentation (R2= 0.88, P<0.0001)

for the individual tumors. Next, we used these two parameters to independently compare the

melanin content found in the CM and UM liver metastases. In situ MRI tumor signal intensity

was significantly different between the two melanoma cohorts (P = 0.003) (Figure 1B). Whereas,

70% of CM metastases had hypointense (0) MRI signal, only 25% of UM metastases displayed

this low signal intensity. Conversely, the UM cohort demonstrated a greater frequency of

metastases with hyperintense (2+) MRI signal (UM: 38% vs. CM: 10%). When these same UM




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and CM metastases underwent pathologic examination after resection, we similarly found a

significant difference in their gross pigmentation (P = 0.008) (Figure 1C). CM metastases were

more often visually hypopigmented (0) (CM: 70% vs. UM: 25%) and UM metastases were more

often hyperpigmentated (2+) (UM: 44% vs. CM: 20%). Thus, we concluded that despite having

common lineage from melanin-producing cells, CM and UM metastases displayed significant

differences in their overall melanin content.

Comparison of melanocyte differentiation antigens and MHC expression between CM and

UM liver metastases

To better understand the differences observed in melanin pigmentation between the CM

and UM liver metastases, we next performed immunohistochemistry (IHC) to compare the

cellular expression of proteins associated with melanocyte differentiation. The tumor expression

(% of viable cells and staining intensity) for MART-1, gp100, and tyrosinase were prospectively

assessed by pathologists blinded to the comparative analysis (Figure 2A). Among the CM liver

metastases, we found prominent heterogeneity in cellular MART-1 expression between

individual metastases (interlesional heterogeneity) and within individual metastases (intralesional

heterogeneity). In 20% of CM metastases, MART-1 was either nearly absent (0-5% of tumor

cells) or expressed on a fraction of viable tumor cells (6-50% of tumor cells). Further, only 34%

of the CM metastases displayed strong cellular staining (3+) for MART-1, while the remaining

tumors had weak to intermediate staining (0 to 2+). In contrast, all of the UM liver metastases

displayed homogeneous, diffuse, and strong MART-1 staining (>50% of tumor cells with 3+

staining intensity). When the pattern of MART-1 expression was compared between the CM and

UM liver metastases, we found UM tumors had significantly stronger MART-1 staining intensity

(P<0.0001) and a trend toward a greater percentage of MART-1 stained tumor cells (P=0.08).




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Expression for gp100 was also greater in the UM metastases compared to the CM metastases

based upon percentage of tumor cells stained (P=0.05) and staining intensity (P=0.01). Diffuse

gp100 staining (>50% of tumor cells) was seen in 88% of UM tumors versus only 58% of CM

tumors. Strong staining intensity (3+) for gp100 was found in 63% of UM tumors versus only

30% of CM tumors. Interestingly, tyrosinase expression (% of tumor cells and staining intensity)

was highly variable in both CM and UM metastases and not significantly different between the

melanoma cohorts (P=0.52 and 0.37, respectively).

Next, we compared the expression of major histocompatibility complex (MHC) class I

and II proteins on tumor cells in the CM and UM liver metastases (Figure 2B). For tumor

antigens to be recognized by T cells, antigens must be internally processed into peptides that are

presented on the tumor cell surface by these MHC molecules. We found MHC class I expression

was equally expressed in both cohorts based upon percentage of tumor cells stained (P=0.73) and

staining intensity (P=0.65). Diffuse MHC class I staining (>50% of tumor cells) was observed in

the majority of tumors from both cohorts (CM: 67% vs. UM: 75%) with equally strong staining

intensity (3+) (CM: 63% vs. UM: 56%). In contrast, we found CM tumors had significantly

greater percentage of MHC class II stained tumor cells (P=0.04) and a trend toward stronger

MHC class II staining intensity (P=0.07). MHC class II expression was nearly undetectable (0-

5% of tumor cells) in 88% of UM tumors versus only 55% of CM tumors. In sum, these IHC

studies demonstrated that UM metastases had greater expression of melanocyte lineage antigens

and lower expression of MHC class II molecules when compared to CM metastases.

Comparison of tumor infiltrating lymphocytes found in CM and UM liver metastases

High levels of tumor infiltrating lymphocytes (TIL) have been reported to correlate with

favorable prognoses in a variety of solid organ malignancies (34-36). Specific immunologic




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studies of TIL expanded from CM metastases have found that these infiltrating cells can often

recognize antigens expressed by the tumor (37). Further, the autologous adoptive transfer of such

TIL has shown durable and complete tumor regression in metastatic CM (12). These findings

have provided compelling evidence for the natural immunogenicity of CM metastases. However,

it is unclear whether UM tumors can similarly elicit adaptive immune responses in vivo. To

provide insight, we sought to compare the attributes of TIL found in UM and CM liver

metastases. First, the degree of infiltrating T cells (CD3, CD4, and CD8 staining) and B cells

(CD20 staining) associated with each of the metastases was prospectively assessed by

pathologists blinded to the comparative analysis. From both tumor cohorts, we found significant

heterogeneity in the numbers of peripheral and infiltrating T cells which ranged from no

lymphocytes detected (0) to extensive lymphoid aggregation occupying over 50% of the tumor

field (3+). When the CM and UM metastases were compared, we found no significant

differences in the levels of peripheral and infiltrating CD3+, CD4+, or CD8+ T cells between

the cohorts (Supplementary Figure 2). Further, B cells (CD20+ cells) were undetectable in the

majority of tumors and also not significantly different between the cohorts.

Having observed that the degree of lymphocytic infiltration was similar between the CM

and UM liver metastases, we next sought to assess the phenotypic and functional attributes of the

TIL after ex vivo expansion. Consecutive metastatic liver tumors were procured from 8 CM and

13 UM patients during a shared time period. To account for intra-tumoral heterogeneity that

might influence TIL growth, 24 geographically discrete tumor fragments were freshly dissected

from each of the metastases and placed in culture media containing human IL-2 (3000IU/ml).

After approximately 2 weeks of culturing, we found that the percentage of tumor fragments that

could successfully generate TIL were equivalent between the CM and UM tumors (95% vs. 94%,




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respectively). Each of these independently expanded TIL cultures were then assessed by flow

cytometry to determine their percentage of CD8+ and CD4+ T cells. We observed a significant

difference between the CM and UM liver metastases in the ratio of these T cell subsets (Figure

3A). The TIL cultures from 88% of the CM metastases (7 of 8 CM metastases) were composed

predominantly of CD8+ T cells. In contrast, only 23% of UM metastases (3 of 13 UM

metastases) gave rise to CD8+ enriched TIL. Cumulatively, the mean percentage of CD8+ T

cells in the CM derived TIL cultures was significantly greater than in the UM derived TIL (CM:

71% vs. UM: 42%, P<0.0001) (Figure 3B). Conversely, UM derived TIL cultures possessed a

greater mean percentage of CD4+ cells when compared with the CM derived TIL cultures (UM:

49% vs. CM: 21%, P<0.0001) (Figure 3B).

Next, we compared the anti-tumor reactivity of the individual CM and UM TIL cultures

by overnight co-culture with tumor digests of their respective parental tumors which had been

freshly cryopreserved at the time of surgical procurement. Reactive TIL cultures were defined as

having tumor induced IFN-γ production >100 pg/ml and twice the background of unstimulated

TIL and tumor digest alone. The autologous anti-tumor reactivity for each of the TIL cultures

from their respective metastases are shown in Figure 3C. The TIL cultures from 88% of the CM

metastases (7 of 8 CM metastases) demonstrated mean tumor specific IFN-γ production >100

pg/ml. In contrast, 46% of UM metastases (6 of 13 UM metastases) had mean reactivity above

this threshold. Cumulatively, CM derived TIL cultures produced higher mean levels of IFN-γ in

response to autologous tumor digest when compared to UM derived TIL cultures (CM: 1044

pg/ml vs. UM: 209 pg/ml, P<0.0001) (Figure 3D). Interestingly, however, we identified

individual TIL cultures from 46% of UM metastases (6 of 13 UM metastases) (L-UM 3b, 5, 7, 8,

12, and14), with IFN-γ production which was comparable in magnitude to the responses




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identified from CM TIL (Figure 3C). Thus, although specific autologous anti-tumor T cell

responses were more prevalent among the CM liver metastases, there was a subset of UM tumors

that could also elicit strong tumor reactive T cell responses.

Metastasis hypopigmentation identifies an immunogenic subset of uveal melanoma

Having found that a subset of UM metastases could naturally elicit auto-reactive TIL

responses, we next sought to determine if there was a clinically relevant means to prospectively

identify UM patients who harbored these immunogenic tumors. Since the majority of CM

metastases possessed TIL with autologous tumor reactivity, we postulated that similar adaptive T

cell responses might preferentially be found in UM metastases with attributes akin to CM

tumors. Pre-operative MRI and gross pathologic examination had demonstrated that the majority

of CM liver metastases lacked melanin pigmentation (Figure 1B). Thus, we investigated whether

the in situ melanin content of UM metastases, as determined by pre-operative MRI imaging,

might correlate with the subsequent growth of auto-reactive TIL populations. The liver

metastases from 13 consecutive UM patients (described in Figure 3C) underwent stratification

based upon their pre-operative in situ radiographic attributes. MRI signal intensity scores

identified four metastases as hyperpigmented (2+) (L-UM 1b, 4, 9, and10), five metastases as

mixed pigmented (1+) (L-UM 3b, 5, 6, 11, and 13), and four metastases as hypopigmented (0)

(L-UM 7, 8, 12, 14). Next, the IFN-γ responses from each of the TIL cultures derived from these

metastases were assessed based upon the MRI characteristics of their parental tumors (Figure 4).

We found that hyperpigmented metastases (2+ MRI signal) uniformly gave rise to TIL cultures

(n=96) with low anti-tumor IFN-γ production (mean: 35pg/ml) that did not exceed background

control levels. In contrast, the mixed pigmented metastases (1+ MRI signal) generated TIL

cultures (n=111) with significantly greater IFN-γ production (mean IFN-γ: 194 pg/ml) (Mixed




21

vs. Hyper P<0.0001); while the hypopigmented metastases (0 MRI signal) were notable for

producing TIL cultures (n=87) with the highest anti-tumor reactivity (mean IFN-γ: 419 pg/ml)

(Hypo vs. Hyper and Mixed, P<0.0001, respectively). Thus, we concluded that low to absent

levels of in situ melanin pigmentation based upon pre-operative clinical MRI could identify a

subset of UM metastases capable of eliciting potent immunogenic TIL responses. In contrast,

UM metastases with high levels of melanin pigmentation identified a non-immunogenic group of

tumors.

Comparison of tumor mutational profile between CM and UM metastases

Although normal differentiation antigens are common targets for endogenous T cells in

melanoma patients, recent studies have demonstrated that unique somatic mutations expressed by

tumors can also elicit autologous T cell responses (37-39). Further, comparative whole exome

sequencing (WES) has revealed sun exposed CM tumors to have the highest number of somatic

mutations among common malignancies (40). These observations have fostered the theory that

the unique responsiveness of metastatic CM to a variety of immunotherapy approaches is a direct

consequence of endogenous immune responses against neo-epitopes encoded by these large

numbers of mutations. Thus, we next sought to determine if the identified subset of

immunogenic UM metastases also harbored a greater mutational load that might explain their

enhanced T cell recognition. Previously, it has been reported that sun-shielded melanomas,

including UM, have far fewer non-synonymous mutations when directly compared with sun-

exposed CM tumors (17). However, these analyses were based upon a limited number of UM

samples which included a mixture of primary and metastatic tumors. Thus, we first sought to

better determine the frequency and characteristics of the non-synonymous mutations occurring in

CM and UM metastases. To provide adequate sample numbers for this analysis we obtained




22

WES data for 278 CM metastases via The Cancer Genome Atlas (TCGA) data portal and

compared these against data from 14 UM metastases from our cohort. Of note, since the TCGA

database does not denote the anatomic site of the metastases, the CM data represents metastases

from a variety of sites. Protein-altering somatic point mutations for each tumor were determined

using a common analytical workflow based upon comparison to matched germline DNA. We

found CM metastases had a broad range in mutation number (range: 6-31,250) when compared

to UM metastases (range: 15-168). Further, as a group, CM metastases had significantly more

somatic mutations when compared to UM metastases (median counts; CM: 282 vs. UM: 73,

P<0.0001) (Figure 5A).

Next, we compared the tumor cohorts for the frequency of prototypic melanoma

associated oncogenic driver mutations including, BRAF, GNAQ and GNA11 (Figure 5B). We

found BRAF mutations in 53% of the CM metastases (n=278). However, BRAF was not mutated

in any of the UM tumors (n=22); (BRAF mutation frequency; CM vs. UM metastases,

P<0.0001). In contrast, activating mutations in either of the homologous genes, GNAQ or

GNA11, were identified in 91% of the UM metastases, but in only 5% of the CM metastases;

(GNAQ/GNA11 mutation frequency; CM vs. UM metastases, P<0.0001).

Finally, we investigated in 12 UM patients whether the mutational frequency identified in

their metastases correlated with the autologous anti-tumor reactivity of their respectively derived

TIL cultures (n~24 cultures/tumor). When the tumor induced IFN-γ production from each of the

TIL cultures was assessed against the number of non-synonymous mutations identified in their

respective parental tumors, we found no correlation between the parameters (Figure 5C).




23

DISCUSSION The last 30 years has provided substantial evidence that the human immune system can

naturally generate potent immunologic responses against tumor antigens expressed by metastatic

cutaneous melanoma (8). Cancer regression can now be achieved in patients with metastatic CM

with mechanistically diverse forms of immunotherapy that augment naturally existing tumor

specific T cell responses (9-12). However, the role of these immune based therapies for the

treatment of metastatic uveal melanoma patients remains unclear. Patients with UM are

frequently excluded from metastatic melanoma immunotherapy clinical trials because UM is

generally thought to be a non-immunogenic form of melanoma (13-16). However, there have not

been formal comparative studies performed directly upon UM and CM metastases to accurately

assess their relative immunogenicity. In this study, we compared the tumor antigen expression,

tumor mutational load, and endogenous anti-tumor immunologic reactivity found in fresh

surgically resected UM and CM metastases. By defining the tumor specific immune responses

that are naturally found in these metastases, we sought to provide insight into the role for

immune based therapies for the management of UM patients. We previously reported that

melanoma metastases demonstrate significant heterogeneity in tumor antigen expression and

lymphocytic infiltrate based upon their anatomic location in the body (6). Thus, to avoid

potential site-specific bias in the current study, we focused our comparative analysis selectively

upon liver metastases resected from UM and CM patients. Our findings revealed that despite

having common melanocytic lineage, UM and CM liver metastases were highly dichotomous in

their melanin content, tumor differentiation antigen expression, and somatic mutational profile.

The majority of CM liver metastases lacked gross melanin pigmentation, whereas UM liver

metastases were more commonly hyperpigmented in appearance. In support of this observation,

immunohistochemical profiling revealed that CM metastases had lower cellular expression of




24

proteins associated with melanocyte differentiation, including MART-1 and gp100. Further, we

found significant differences in the overall somatic mutational profile between CM and UM liver

metastases. Comparative whole exomic sequencing revealed CM metastases had significantly

greater mutational burden compared to UM metastases with the melanoma variants also

possessing quite different oncogenic driver mutations of the MAPK pathway. Similar to previous

reports (41-43), nearly all of the UM metastases had GNAQ and GNA11 mutations, while CM

metastases commonly had BRAF mutations. Collectively, these comparative studies demonstrate

CM metastases to be far more de-differentiated from their melanocytic origin when compared to

UM metastases in terms of their mutational profile, tumor antigen expression, and gross melanin

pigmentation.

When endogenous immune responses in these highly divergent forms of melanoma were

characterized, we further identified marked differences in the phenotype and anti-tumor

reactivity of their respective infiltrating lymphocytes. CM TIL were predominantly composed of

CD8+ T cells, while UM TIL were CD4+ dominant. Reactivity against autologous tumor was

significantly greater in CM TIL compared to UM TIL. However, we identified TIL from a subset

of UM patients which had robust anti-tumor reactivity that was comparable in magnitude to that

of CM TIL. The identification of this immunogenic group of UM metastasis has not been

previously reported and thus, has fostered our interest in determining the specific antigenic

targets that are recognized by these UM derived TIL. Interestingly, the level of in situ melanin

pigmentation found in parental tumors strongly correlated with the generation of tumor reactive

UM TIL. Hyperpigmented UM metastases generated TIL cultures with poor tumor reactivity,

while the metastases that lacked pigmentation produced the most reactive TIL cultures. Although

the precise mechanism underlying the relationship between tumor pigmentation and TIL




25

reactivity is not completely understood, the loss of pigment proteins by metastatic tumor cells is

thought to be driven by the stochastic genetic instability of tumor cells combined with the non-

stochastic selective pressures of the host immune system (immunoediting)(44-48). Animal

models have demonstrated the development of vitiligo and the loss of tumor pigment proteins

through the action of antigen specific CD8+ T cells (49). Further, it is well known that human

CM TIL frequently possess T cells specific for melanocyte pigment proteins, such as MART-1

and gp100 (50). Thus, in the current study, we hypothesize that the loss of tumor pigmentation in

this subset of UM metastases could signify the presence of a vigorous immune response targeting

pigment antigens. However, beyond the targeting of these normal differentiation antigens, recent

analyses have found that unique somatic mutations expressed by tumors can generate neo-

epitopes that also elicit robust autologous T cell responses (37-39). Although our study found no

correlation between the mutational frequencies identified in UM metastases and the autologous

anti-tumor reactivity of their respectively derived TIL cultures, there may still be individual

mutations that are recognized. Thus, we have begun studies to assess the tumor reactive UM TIL

for recognition of both non-mutated, as well as, mutated antigen targets.

Although not completely validated as a clinical biomarker, the MRI assessment of

melanin content in UM metastases was found in this study to accurately identify tumors that can

elicit a strong endogenous immune response. We are, thus, interested in determining whether

immune based therapies may be more effective in the subset of UM patients who harbor these

unique immunogenic tumors. To help address these questions, we are conducting the first in-

human adoptive T cell transfer trial dedicated to patients with metastatic UM (NCT01814046).

In this phase II study, patients with metastatic UM undergo surgical metastasectomy to procure

tumor tissue for TIL generation. The expanded lymphocytes are then adoptively transferred back




26

into the host in conjunction with a nonmyeloablative lymphodepleting regimen. The primary

endpoint of this study is to define the objective response rate of TIL immunotherapy in patients

with metastatic UM. The results of this trial should provide valuable insight into the role of

immune based therapies for the treatment of metastatic uveal melanoma.




27

Acknowledgements

We thank the Surgery Branch cell production facility and the immunotherapy clinical and

support staff for their contributions. We thank Li Jia for assistance in bioinformatics analysis.

The Prospective Procurement of Solid Tumor Tissue to Identify Novel Therapeutic study was

supported by the Intramural Research Program of the National Cancer Institute, National

Institutes of Health, Department of Health and Human Services. Whole exome raw data was

uploaded to the NIH database for Genotypes and Phenotypes (dbGaP) under accession number

phs001003.v1.p1.

.




28

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32

Table 1. Patient demographics and procurement of liver metastases

Uveal Melanoma

Cutaneous Melanoma

Abbreviations: Bn, Bone; Li, Liver; LN, Lymph Node; Lu, Lung; Om, Omentum; Panc, Pancreas; Per, Peritoneum; Spl, Spleen; ST, Soft tissue Research.

on August 13, 2020. © 2015 American Association for Cancerclincancerres.aacrjournals.org Downloaded from



33

Table 2. Comparison of CM and UM patients who underwent liver metastasectomy




34

FIGURE LEGENDS

Figure 1. UM liver metastases have greater melanin pigmentation when compared to CM

liver metastases. (A) Illustrative examples demonstrating the correlation between pre-operative

in situ MRI intensity scoring and post-operative gross pathologic scoring of pigmentation in

selected melanoma liver metastases. Arrows in the left panel indicate the tumors that underwent

metastasectomy; right panel displays photos of the resected tumors. (B) Comparison of CM and

UM liver metastases based upon MRI tumor signal intensity score and (C) gross pathologic

pigmentation score. Numbers within the bubbles denote the percentage of tumors in the specified

cohort with the indicated score. Bubble diameter is proportionate to these percentages. Statistical

comparisons between CM and UM cohorts were performed with the Mann-Whitney test.

Figure 2. UM liver metastases have greater expression of melanocyte differentiation

antigens and lower MHC class II compared to CM liver metastases. Comparison of CM and

UM liver metastases based upon immunohistochemical staining of (A) prototypic melanocyte

differentiation antigens and (B) MHC class I and II molecules. Upper panels demonstrate

expression based upon percentage of viable tumor cells within individual metastases that stain

with the indicated marker; lower panels demonstrate expression based upon tumor cell staining

intensity. Numbers within the bubbles denote the percentage of tumors in the specified cohort

with the indicated marker expression. Bubble diameter is proportionate to these percentages.

Statistical comparisons between CM and UM cohorts were performed with the Mann-Whitney

test.

Figure 3. Phenotypic and functional comparison of TIL cultures derived from CM and UM

liver metastases. (A) Mean percentage of CD8+ (filled box) and CD4+ (open box) T cells found

in TIL cultures derived from individual CM and UM liver metastases. T cell subset frequency as




35

assessed by flow cytometry and CD3 gating. Box denotes the mean of approximately 24

individual TIL cultures with error bars indicating SEM. (B) Cumulative comparison of CM

versus UM derived TIL cultures based upon their percentage of CD8+ and CD4+ T cells. Bars

demonstrate the mean with error bars indicating SEM. (C) Autologous tumor reactivity of

individual TIL cultures derived from CM and UM liver metastases. Each dot represents the IFN-

γ production of a single TIL culture in response to overnight co-culture with autologous tumor

digest minus the background cytokine levels of unstimulated TIL and tumor digest alone. Bar

represents the mean of approximately 24 TIL cultures. Dashed line marks 100pg/ml of IFN- γ on

the y-axis. (D) Cumulative comparison of the anti-tumor reactivity of CM and UM derived TIL

cultures based upon production of IFN-γ in response to autologous tumor digest. Bars

demonstrate the mean with error bars indicating SEM. Statistical comparisons between CM and

UM cohorts were performed with the Student’s t test.

Figure 4. Metastasis hypopigmentation identifies an immunogenic subset of uveal

melanoma. Association between the pre-operative melanin content of UM metastases and the

autologous anti-tumor reactivity of their derived TIL cultures. UM TIL cultures (n~24) were

established from individual liver metastases from 13 UM patients. The cultures were stratified

into cohorts based upon the melanin pigmentation status of their parental tumors as assessed by

pre-operative in situ MRI signal intensity scoring. Each dot represents the IFN-γ production of a

single UM TIL culture in response to overnight co-culture with autologous tumor digest minus

the background cytokine levels of unstimulated TIL and tumor digest alone. Statistical

comparisons between the stratified pigmentation cohorts were performed with the Student’s t

test.




36

Figure 5. Tumor mutational profiles of CM and UM metastases (A) Comparison of the

number of non-synonymous mutations identified in CM and UM metastases. Box extends from

25th to 75th percentile, line through the box indicates median, and bars extend from the smallest

to largest values. Statistical comparison between CM and UM cohorts was performed with the

Student’s t test. (B) Frequency of BRAF, GNAQ and GNA11 mutations identified in CM and UM

metastases. Statistical comparison of oncogene frequency between CM and UM were performed

with Fisher’s exact test. (C) Correlation between the number of non-synonymous mutations

identified in UM metastases and the autologous anti-tumor reactivity of their derived TIL

cultures. Data represents UM TIL cultures (n~24) established from metastases from 12 UM

patients. Each dot plots the mutation frequency of the parental tumor (x-axis) versus the IFN-γ

production of the derived UM TIL cultures in response to overnight co-culture with autologous

tumor digest minus the background cytokine levels of unstimulated TIL and tumor digest alone

(y-axis). Linear regression analysis was used to derive R2 values.




70

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L-CM29

L-CM30

L-CM31

L-CM32

L-CM33

L-CM34

L-CM35

L-UM1b

L-UM3b

L-UM4

L-UM5

L-UM6

L-UM7

L-UM8

L-UM9

L-UM10

L-UM11

L-UM12

L-UM13

L-UM14

10

1

10

2

10

3

10

4

10

5

<

L-CM28

L-CM29

L-CM30

L-CM31

L-CM32

L-CM33

L-CM34

L-CM35

L-UM1b

L-UM3b

L-UM4

L-UM5

L-UM6

L-UM7

L-UM8

L-UM9

L-UM10

L-UM11

L-UM12

L-UM13

L-UM14

0

20

40

60

80

10

0

L-CM28

L-CM29

L-CM30

L-CM31

L-CM32

L-CM33

L-CM34

L-CM35

L-UM1b

L-UM3b

L-UM4

L-UM5

L-UM6

L-UM7

L-UM8

L-UM9

L-UM10

L-UM11

L-UM12

L-UM13

L-UM14

0

20

40

60

80

10

0C

M T

IL C

ult

ure

sU

M T

IL C

ult

ure

s

% of cells with marker

CD

8C

D4

CD

8C

D4

CM28

CM29

CM30

CM31

CM32

CM33

CM34

CM35

UM1b

UM3b

UM4

UM5

UM6

UM7

UM8

UM9

UM10

UM11

UM12

UM13

UM14

0

20

40

60

80

10

0C

M2

8

UM

4

CM28

CM29

CM30

CM31

CM32

CM33

CM34

CM35

UM1b

UM3b

UM4

UM5

UM6

UM7

UM8

UM9

UM10

UM11

UM12

UM13

UM14

0

20

40

60

80

10

0C

M3

2

UM

3b

CD

8C

D4

CD

8C

D4

CM28

CM29

CM30

CM31

CM32

CM33

CM34

CM35

UM1b

UM3b

UM4

UM5

UM6

UM7

UM8

UM9

UM10

UM11

UM12

UM13

UM14

0

20

40

60

80

10

0C

M2

8

UM

4

CM28

CM29

CM30

CM31

CM32

CM33

CM34

CM35

UM1b

UM3b

UM4

UM5

UM6

UM7

UM8

UM9

UM10

UM11

UM12

UM13

UM14 0

20

40

60

80

10

0C

M3

2

UM

3b

IFN-g(pg/ml)

Liv

er

Meta

sta

ses I

D#

0

50

0

10

00

15

00

P<

0.0

00

1

IFN-g(pg/ml)

CM

UM

0

20

40

60

80

10

0

Su

mm

ary

of

TIL

cu

ltu

re p

hen

oty

pe

CD

8

P<

0.0

00

1

CD

4

P<

0.0

00

1

CM

UM

CM

UM

Su

mm

ary

of

TIL

cu

ltu

re r

eacti

vit

y a

gain

st

au

tolo

go

us f

resh

tu

mo

r

% of cells with marker

Fig

ure

3.

A.

B.

C.

D.




Figure 4.

Association between in situ pigmentation status of

UM metastases and TIL culture reactivity

IFN

-g(p

g/m

l)

0

Hypo

(n=87)

419

224

10-2467

1+

Mixed

(n=111)

194

49

10-1706

MRI pigmentation

status:

TIL Cultures:

Mean:

Median:

Range:

2+

Hyper

(n=96)

35

10

10-306

Hy

po

Mix

ed

Hy

pe

r

0

1 0 0 0

2 0 0 0

3 0 0 0P=0.0001

P<0.0001

P<0.0001

IFN-g




CM

(n=278)

590

282

6-31250

UM

(n=14)

82

73

15-168

P<0.0001

Nu

mb

er

of

no

n-s

yn

on

ym

ou

s

mu

tati

on

s

1 0 0

1 0 1

1 0 2

1 0 3

1 0 4

1 0 5

C u ta n e o u s

Mean:

Median:

Range:

GNA1150%

(n=11)GNAQ41%(n=9)

Neither9%

(n=2)

No BRAF100%(n=22)

CM (n=278)

BRAF53%

(n=146)

No BRAF47%

(n=132)

GNA112%

(n=6)

GNAQ3%

(n=8)

Neither95%

(n=264)

UM (n=22)

BR

AF

mu

tati

on

sG

NA

Q/1

1 m

uta

tio

ns

CM vs. UM

P<0.0001

P<0.0001

Tumor Mutational Analysis

A. B.IF

N-g

(pg

/ml)

Number of non-synonymous

mutations

R2 = 0.01

P = 0.76

Correlation between UM metastasis

mutational load and TIL culture reactivity

0 5 0 1 0 0 1 5 0 2 0 0

0

1 0 0 0

2 0 0 0

3 0 0 0

Figure 5.

C.




Published OnlineFirst December 28, 2015.Clin Cancer Res Luke D. Rothermel, Arvind Sabesan, Daniel J. Stephens, et al. melanomaIdentification of an immunogenic subset of metastatic uveal

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Documents

Identification of an immunogenic subset of metastatic uveal melanoma · Identification of an immunogenic subset of metastatic uveal melanoma Luke D. Rothermel*1, Arvind C. Sabesan*1,

Identification of an immunogenic subset of metastatic uveal melanoma · Identification of an immunogenic subset of metastatic uveal melanoma Luke D. Rothermel1, Arvind C. Sabesan1,